The Factor VIII complex has two distinct biologic functions coagulant activity and a role in primary hemostatis. The analysis of Factor VIII deficiency diseases, classic hemophilia and von Willebrand's disease, have contributed to the understanding that Factor VIII is a complex of two components. The Factor VIII:c procoagulant protein (antihemophilic factor) and the Factor VIII related antigen (von Willebrand factor, VWF) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions.
The Factor VIII:c molecule is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of Factor X activation by Factor IXa, eventually leading to the formation of the fibrin clot. Deficiency of Factor VIII:c (classic hemophilia) is an X-linked chromosomal disorder that has been a major source of hemorrhagic morbidity and mortality in affected males. Treatment usually consists of frequent transfusions with blood products. The latter has resulted in a high incidence of infectious complications (such as various forms of hepatitis and acquired immunodeficiency disease) in the hemophiliac population.
The VWF molecule is an adhesive glycoprotein that plays a central role in platelet agglutination. It serves as a carrier for Factor VIII:c in plasma and facilitates platelet-vessel wall interactions. Discrete domains of VWF which bind to platelet receptor sites on glycoprotein 1b and on the glycoprotein IIb-IIIa complex, as well as binding sites on collagen have been noted. VWF is made up of multiple, probably identical, subunits each of 230,000 daltons. VWF is synthesized in endothelial cells and megakaryocytes. In the plasma it exists as high molecular weight multimers ranging from 5.times.10.sup.5 to 10.sup.7 daltons. The Von Willebrand factor contains 5-6% complex carbohydrate, which appears important in the molecules ability to bind platelets. A variety of abnormalities in VWF activity can result in Von Willebrand's disease. The disorder is generally inherited in autosomal dominant fashion and may affect as many as one in 2000 individuals. Mild forms of the disease frequently go undiagnosed, whereas severely affected patients may require frequent blood product support with its associated risks.
Recently, the isolation of the genes for both factor VIII:c and Von Willebrand factor have made feasible the production of recombinant factor VIII:c and VWF preparations which are essentially free of contaminating viruses (Toole et al. 1984, Wood et al. 1984, Lynch et al. Cell 41:49-56 1985, Ginsberg et al. Science 228:1401-1406 1985). The production of Factor VIII:c through recombinant DNA technology has been achieved utilizing mammalian cells as a recipient of DNA encoding Factor VIII:c contained in appropriate expression sectors. One primary concern for the synthesis of a recombinant Factor VIII:c is its stability in the absence of the associated VWF protein Evidence to date indicates either that the VWF may have a stabilizing effect on the Factor VIII:c in plasma, or that the VWF can ellicit the release from storage depots or stimulate the synthesis and/or secretion of Factor VIII:c (Weiss, H. J. et al. 1977, J. Clin. Invest. 60: 390-404).
It has now been surprisingly discovered that suitably engineered mammalian cells produce significant levels of stable Factor VIII only in the presence of media containing a hydrophobic substance such as certain phospholipids or VWF, as is the case when the cell culture media contains serum. The Factor VIII:c synthesized in the absence of VWF or suitable phospholipids exhibits dramatic instability. VWF has been identified as one of the stabilizing components in serum. The gene for VWF has been expressed in a mammalian host cell to derive a protein which has the capability to stabilize Factor VIII:c expresssed in cells which are grown in the absence of serum. The expression of both Factor VIII:c (or analogs thereof) and VWF in mammalian cells allows for the production in improved yield of stable recombinant Factor VIII (or analogs thereof) in cells propagated in the substantial absence of serum.